Inhibition of arterivirus RNA synthesis by cyclophilin inhibitors is counteracted by mutations in replicase transmembrane subunits

Adriaan H. de Wilde; A. Linda Boomaars-van der Zanden; Anja W. M. de Jong; Montserrat Barcéna; Eric J. Snijder; Clara C. Posthuma

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/587261 doi: bioRxiv preprint phenomenon that was independent of EAV infection as it was also observed in CsA-treated mock-335 infected Huh7 cells (compare Figs. 5A-C and 5D-F). Taken together, these observations strongly 336 suggest that CsA treatment interferes with a very early stage of arterivirus infection, i.e. prior to or 337 during RO formation. Apparently, adaptive mutations in nsp5 can compensate for the detrimental 338 effects of CsA treatment, which may either target one of the viral players directly or affect a host 339 factor involved in arterivirus replication, like cyclophilin A (see above). For this purpose, BHK-21 cells were infected with rEAV wt or rEAV QYA and viral RNA synthesis was 355 assayed by metabolic labelling with [ 3 H]uridine from 6.5 to 7.5 h p.i. (Fig. 7A, B ). Cells were also 356 treated with dactinomycin to block cellular transcription. [ 3 H]-uridine incorporation into viral RNA 357 was quantified by isolation of intracellular RNA and liquid scintillation counting (Fig. 7A ). Labeled 358 viral RNA was also visualized by agarose gel electrophoresis and fluorography (Fig. 7B ). This 359 revealed that RNA synthesis by the mutant virus was approximately 40% of wt virus activity, which is 360 in line with the observed delayed growth kinetics and reduced titers of the mutant. However, in the 361 All rights reserved. No reuse allowed without permission.